The proposed research aims to investigate the genetic and biochemical mechanisms of gene regulation in Streptococcus mutans through the irvA-dependent pathway. The goals of this project will be achieved in three aims by: 1) Analyzing the mechanism of irvA induction 2) Identifying the IrvA regulon 3) Characterizing the genetic regulators that comprise the irvA-dependent pathway. 1) To analyze the mechanism of irvA induction, we will perform detailed biochemical studies of key residues of SMu1275 to determine how its endogenous autocleavage capacity can modulate its ability to control irvA expression. 2) To identify the IrvA regulon, we will employ chromatin immunoprecipitation and microarray (ChIP-chip). Using specially designed Affymetrix Genechips(R), we will be able to detect most or all of the IrvA binding sites in vivo. Transcription analysis of these genes will determine the role of IrvA as a transcription regulator of genes associated with stress and virulence related phenotypes. 3) To characterize the genetic regulators of the irvA-dependent pathway, we will first identify the genes that are capable of inducing irvA. Genetic analyses will then examine the role of irvA as a mediator of stress and virulence related phenotypes in these strains. Finally, genes coregulated with the irvA-dependent pathway will be identified. The goal of this project is to use the irvA-dependent pathway as a model to examine the connection between stress adaptation and the regulation of important virulence factors of Streptococcus mutans. Since the long-term persistence of S. mutans requires the coordination of stress response mechanisms and virulence factor gene expression, these data may yield new insights into S. mutans cariogenicity. PUBLIC HEALTH RELEVANCE: Dental caries is the most common human bacterial disease and is largely correlated with the overgrowth of Streptococcus mutans. Since this organism's ability to cause disease requires careful coordination of its stress response and virulence, these studies may yield novel treatment strategies to control the pathogenesis of S. mutans.